Fig. 7

S-nitrosylation of VEGFD affects its secretion. A-B HEK-293T cells were transfected with vector or VEGFD-expressing construct, cultured for 24 h, and then treated with L-NAME or GSNO for further 24 h. ELISA assays detects secreted VEGFD levels in the culture supernatants. C-D HEK-293T cells were transfected with vector (V) or HA-VEGFD (VD), pretreated with ACTD for 6 h, and then treated with GSNO at 50µM or DTT at 10µM for further 24 h. ELISA assays detects secreted VEGFD levels in the culture supernatants. (E)Western blot analyses for VEGFD in the culture supernatants and 293T cells transfected with vector, HA-VEGFD (WT) or HA-VEGFD(C277S) mutant and then cultured for 48 h. F HEK-293T cells were transfected with WT or C277S mutant, ELISA assays detected secreted VEGFD in the culture supernatants. G NCI-H1975 cells were transfected with vector, WT or C277S mutant and then cultured for 24 h, media was collected to treat HUVEC cells for 24 h, and then HUVEC cells were harvested for qPCR. H Schematic diagram for the proteolytic cleavages of VEGFD by proteases. I ELISA assays for the culture supernatants of 293T cells at 48 h post co-transfection with WT/C277S and PIA/PC-5/PC-7. J Western blot analyses in 293T cells at 24 h post co-transfection with WT/C277S and PC-7. Data in (E) and (J) are representative of three independent experiments. and data in (A), (B), (C), (D), (F), (G), and (I) represent the mean ± SEM of triplicate samples. * p < 0.05, **p < 0.01. Student’s t test